Sensitive aptasensing of tobramycin through a rational design of catalytic hairpin assembly and hybridization chain reaction amplification monomers for CRISPR/Cas12a activation

The catalytic hairpin assembly (CHA) and hybridization chain reaction amplification (HCR) are enzyme-free isothermal DNA amplification methods based on the self-assembly of hairpin monomers. Recently, CRISPR/Cas12a-based biosensors in combination with CHA or HCR signal amplification have shown promising performance. Herein, several design strategies for hairpin monomers in CHA and HCR were evaluated in the context of CRISPR/Cas12a-based biosensor construction. The SL-HCR strategy, in which the CRISPR/Cas12a target strand is blocked in the loop of one hairpin monomer DNA and released in the duplex HCR products, demonstrated superior performance in terms of a low background signal, wide linear detection range, and high signal-to-noise ratio. With the assistance of an aptamer-containing probe, a highly sensitive aptasensor was constructed for tobramycin detection, whereby the SL-HCR served the function of signal amplification, whereas the CRISPR/Cas12a system acted to cleave the FQ probes, thereby resulting in the production of a fluorescent signal. After optimization, the aptasensor enables linear detection of tobramycin concentrations ranging from 125 pM to 2500 nM, with a limit of detection (LOD) of 92.87 pM. Moreover, the aptasensor was utilized to detect tobramycin in beef and milk samples, yielding satisfactory results. The assay is concise and cost-effective due to the absence of nanomaterial DNA labeling and magnetic separation procedures. Furthermore, the entire detection workflow operates under isothermal conditions, which makes it suitable for use in food safety control and environmental monitoring. In addition, the results presented here may shed new light on the design of CRISPR/Cas12a-based biosensors in combination with CHA or HCR.